Spinach line SMBS013-1209F

ABSTRACT

The invention provides seeds and plants of spinach line SMBS013-1209F. The invention thus relates to the plants, seeds, plant parts, and tissue cultures of spinach line SMBS013-1209F and to methods for producing a spinach plant produced by crossing such plants with themselves or with another plant, such as a spinach plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to plants, seeds, plant parts, and tissue cultures of spinach line SMBS013-1209F comprising introduced beneficial or desirable traits.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, morespecifically, to the development of spinach line SMBS013-1209F.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits ina single variety. Such desirable traits may include any trait deemedbeneficial or desirable by a grower or consumer, including greateryield, resistance to insects or disease, tolerance to environmentalstress, and nutritional value.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all genetic loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different genotypes produces auniform population of hybrid plants that are heterozygous for manygenetic loci. Conversely, a cross of two plants each heterozygous at anumber of loci produces a population of hybrid plants that differgenetically and are not uniform. The resulting non-uniformity makesperformance unpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines and hybrids derivedtherefrom are developed by selfing and selection of desired phenotypes.The new lines and hybrids are evaluated to determine which of those havecommercial potential.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a spinach plant of spinachline SMBS013-1209F. Also provided are spinach plants having all thephysiological and morphological characteristics of such a plant. Partsof these spinach plants are also provided, for example, includingpollen, an ovule, an embryo, a seed, a scion, a rootstock, a fruit, anda cell of the plant.

In another aspect of the invention, a plant of spinach lineSMBS013-1209F comprising an added heritable trait is provided. Theheritable trait may comprise a genetic locus that is, for example, adominant or recessive allele. In one embodiment of the invention, aplant of spinach line SMBS013-1209F is defined as comprising a singlelocus conversion. In specific embodiments of the invention, an addedgenetic locus confers one or more traits such as, for example, herbicidetolerance, insect resistance, disease resistance, and modifiedcarbohydrate metabolism. In further embodiments, the trait may beconferred by a naturally occurring gene introduced into the genome of aline by backcrossing, a natural or induced mutation, or a transgeneintroduced through genetic transformation techniques into the plant or aprogenitor of any previous generation thereof. When introduced throughtransformation, a genetic locus may comprise one or more genesintegrated at a single chromosomal location.

In some embodiments, a single locus conversion includes one or moresite-specific changes to the plant genome, such as, without limitation,one or more nucleotide modifications, deletions, or insertions. A singlelocus may comprise one or more genes or nucleotides integrated ormutated at a single chromosomal location. In one embodiment, a singlelocus conversion may be introduced by a genetic engineering technique,methods of which include, for example, genome editing with engineerednucleases (GEEN). Engineered nucleases include, but are not limited to,Cas endonucleases; zinc finger nucleases (ZFNs); transcriptionactivator-like effector nucleases (TALENs); engineered meganucleases,also known as homing endonucleases; and other endonucleases for DNA orRNA-guided genome editing that are well-known to the skilled artisan.

The invention also concerns the seed of spinach line SMBS013-1209F. Theseed of the invention may be provided as an essentially homogeneouspopulation of seed of spinach line SMBS013-1209F. Essentiallyhomogeneous populations of seed are generally free from substantialnumbers of other seed. Therefore, seed of spinach line SMBS013-1209F maybe defined as forming at least about 97% of the total seed, including atleast about 98%, 99%, or more of the seed. The seed population may beseparately grown to provide an essentially homogeneous population ofspinach plants designated SMBS013-1209F.

In yet another aspect of the invention, a tissue culture of regenerablecells of a spinach plant of line SMBS013-1209F is provided. The tissueculture will preferably be capable of regenerating spinach plantscapable of expressing all of the physiological and morphologicalcharacteristics of the starting plant and of regenerating plants havingsubstantially the same genotype as the starting plant. Examples of someof the physiological and morphological characteristics of spinach lineSMBS013-1209F include those traits set forth in the table herein. Theregenerable cells in such tissue cultures may be derived, for example,from embryos, meristems, cotyledons, pollen, leaves, anthers, roots,root tips, pistils, flowers, seed, and stalks. Still further, thepresent invention provides spinach plants regenerated from a tissueculture of the invention, the plants having all the physiological andmorphological characteristics of spinach line SMBS013-1209F.

In still yet another aspect of the invention, processes are provided forproducing spinach seeds, plants, and fruit, which processes generallycomprise crossing a first parent spinach plant with a second parentspinach plant, wherein at least one of the first or second parent plantsis a plant of spinach line SMBS013-1209F. These processes may be furtherexemplified as processes for preparing hybrid spinach seed or plants,wherein a first spinach plant is crossed with a second spinach plant ofa different, distinct genotype to provide a hybrid that has, as one ofits parents, a plant of spinach line SMBS013-1209F. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent spinach plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent spinach plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the flowers (i.e., killing or removing the pollen).

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent spinach plants. Yet another step comprisesharvesting the seeds from at least one of the parent spinach plants. Theharvested seed can be grown to produce a spinach plant or hybrid spinachplant.

The present invention also provides the spinach seeds and plantsproduced by a process that comprises crossing a first parent spinachplant with a second parent spinach plant, wherein at least one of thefirst or second parent spinach plants is a plant of spinach lineSMBS013-1209F. In one embodiment of the invention, spinach seed andplants produced by the process are first generation (F₁) hybrid spinachseed and plants produced by crossing a plant in accordance with theinvention with another, distinct plant. The present invention furthercontemplates plant parts of such an F₁ hybrid spinach plant, and methodsof use thereof. Therefore, certain exemplary embodiments of theinvention provide an F₁ hybrid spinach plant and seed thereof.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from spinach line SMBS013-1209F, the methodcomprising the steps of: (a) preparing a progeny plant derived fromspinach line SMBS013-1209F, wherein said preparing comprises crossing aplant of spinach line SMBS013-1209F with a second plant; and (b)crossing the progeny plant with itself or a second plant to produce aseed of a progeny plant of a subsequent generation. In furtherembodiments, the method may additionally comprise: (c) growing a progenyplant of a subsequent generation from said seed of a progeny plant of asubsequent generation and crossing the progeny plant of a subsequentgeneration with itself or a second plant; and repeating the steps for anadditional 3-10 generations to produce a plant derived from spinach lineSMBS013-1209F. The plant derived from spinach line SMBS013-1209F may bean inbred line, and the aforementioned repeated crossing steps may bedefined as comprising sufficient inbreeding to produce the inbred line.In the method, it may be desirable to select particular plants resultingfrom step (c) for continued crossing according to steps (b) and (c). Byselecting plants having one or more desirable traits, a plant derivedfrom spinach line SMBS013-1209F is obtained which possesses some of thedesirable traits of the line/hybrid as well as potentially otherselected traits.

In certain embodiments, the present invention provides a method ofproducing food or feed comprising: (a) obtaining a plant of spinach lineSMBS013-1209F, wherein the plant has been cultivated to maturity, and(b) collecting at least one spinach from the plant.

In still yet another aspect of the invention, the genetic complement ofspinach line SMBS013-1209F is provided. The phrase “genetic complement”is used to refer to the aggregate of nucleotide sequences, theexpression of which sequences defines the phenotype of, in the presentcase, a spinach plant, or a cell or tissue of that plant. A geneticcomplement thus represents the genetic makeup of a cell, tissue orplant, and a hybrid genetic complement represents the genetic make-up ofa hybrid cell, tissue or plant. The invention thus provides spinachplant cells that have a genetic complement in accordance with thespinach plant cells disclosed herein, and seeds and plants containingsuch cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that spinach line SMBS013-1209F could be identified byany of the many well-known techniques such as, for example, SimpleSequence Length Polymorphisms (SSLPs) (Williams et al., Nucleic AcidsRes., 1 8:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs),DNA Amplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by spinach plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a spinach plant of the invention with a haploid geneticcomplement of a second spinach plant, preferably, another, distinctspinach plant. In another aspect, the present invention provides aspinach plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

The term “about” is used to indicate that a value includes the standarddeviation of the mean for the device or method being employed todetermine the value. The use of the term “or” in the claims is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternatives are mutually exclusive. When used in conjunctionwith the word “comprising” or other open language in the claims, thewords “a” and “an” denote “one or more,” unless specifically notedotherwise. The terms “comprise,” “have,” and “include” are open-endedlinking verbs. Any forms or tenses of one or more of these verbs, suchas “comprises,” “comprising,” “has,” “having,” “includes,” and“including,” are also open-ended. For example, any method that“comprises,” “has,” or “includes” one or more steps is not limited topossessing only those one or more steps and also covers other unlistedsteps. Similarly, any plant that “comprises,” “has,” or “includes” oneor more traits is not limited to possessing only those one or moretraits and covers other unlisted traits.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds, and derivatives of spinach line SMBS013-1209F. This line showsuniformity and stability within the limits of environmental influencefor the traits described hereinafter. Spinach line SMBS013-1209Fprovides sufficient seed yield. By crossing with a distinct secondplant, uniform F₁ progeny can be obtained.

A. Physiological and Morphological Characteristics of Spinach LineSMBS013-1209F

In accordance with one aspect of the present invention, there areprovided plants having the physiological and morphologicalcharacteristics of spinach line SMBS013-1209F. Descriptions of thephysiological and morphological characteristics of such plants arepresented in the table that follows.

TABLE 1 Physiological and Morphological Characteristics of Spinach LineSMBS013-1209F CHARACTERISTIC SMBS013-1209F SSB-66-1087F Ploidy diploiddiploid Seedling Cotyledon length (mm) 61.5 54.1 length short/mediumshort width (mm) 6.7 6.3 tip pointed pointed color medium green mediumgreen color (RHS Color Chart Value) 146b 146b Leaf (First FoliageLeaves) shape elliptic elliptic base v-shape v-shape tip round-pointedround-pointed margin slightly curled slightly curled upper surface colormedium green medium green color (RHS Color Chart Value) 146a 146a lowersurface color (compared lighter lighter with upper surface) color (RHSColor Chart Value) 146b 146b Maturity growth rate medium slow number ofdays from planting 41 48 to prime market stage Plant (Prime MarketStage) habit flat flat size large large spread (cm) 49 51 height (cm) 119 Leaf (Prime Market Stage) surface smooth smooth anthocyanin colorationof absent absent petioles and veins blade, intensity of green colormedium medium blade blistering weak weak blade lobing absent or veryabsent or very weak weak petiole attitude horizontal horizontal petiolelength medium medium blade attitude horizontal horizontal leaf shapeelliptic elliptic blade shape (excluding basal broad elliptic mediumelliptic lobes) blade, curving of margin recurved recurved blade, shapeof apex rounded rounded blade shape in longitudinal flat flat sectionbase straight straight tip round round margin curled under curled underupper surface color medium green medium green color (RHS Color ChartValue) 146a 146a lower surface color (compared lighter lighter withupper surface) color (RHS Color Chart Value) 146b 146b luster dull dullblade size large medium petiole color light green light green color (RHSColor Chart Value) 144a 144a petiole red pigmentation absent absentpetiole length to the blade (cm) 13.9 9.6 petiole diameter (mm) 11.0 9.9petiole diameter large large Seed Stalk Development start of bolting(10% of plants) late late time of start of bolting (spring late latesown crop, 15% of plants) height of stalk (cm) 81.3 87.8 leaves on stalkof female plant many many proportion of female plants very high veryhigh plants that are female 91-100% 91-100% (percentage) plants that aremale 0-10% 0-10% (percentage) proportion of male plants absent or verylow absent or very low plants that are monoecious 0-10% 0-10%(percentage) proportion of monoecious absent or very low absent or verylow plants Seed surface smooth smooth spines (harvested seed) absentabsent These are typical values. Values may vary due to environment.Values that are substantially equivalent are within the scope of theinvention.

B. Breeding Spinach Plants

In one aspect of the current invention, spinach line SMBS013-1209F maybe crossed with itself or with any second plant. Such methods can beused for propagation of spinach line SMBS013-1209F or can be used toproduce plants that are derived from spinach line SMBS013-1209F. Plantsderived from spinach line SMBS013-1209F may be used, in certainembodiments, for the development of new spinach varieties.

The development of new varieties using one or more starting varieties iswell-known in the art. In accordance with the invention, novel varietiesmay be created by crossing spinach line SMBS013-1209F followed bymultiple generations of breeding according to such well-known methods.New varieties may be created by crossing with any second plant. Inselecting such a second plant to cross for the purpose of developingnovel lines, it may be desired to choose those plants which eitherthemselves exhibit one or more selected desirable characteristics orwhich exhibit the desired characteristic(s) when in hybrid combination.Once initial crosses have been made, inbreeding and selection take placeto produce new varieties. For development of a uniform line, often fiveor more generations of selfing and selection are involved.

Uniform lines of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g., colchicine treatment). Alternatively, haploid embryos may begrown into haploid plants and treated to induce chromosome doubling. Ineither case, fertile homozygous plants are obtained. In accordance withthe invention, any of such techniques may be used in connection with aplant of the invention and progeny thereof to achieve a homozygous line.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny have the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

The plants of the present invention are particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the plants. In selecting a second plant to cross withspinach line SMBS013-1209F for the purpose of developing novel spinachlines, it will typically be preferred to choose those plants whicheither themselves exhibit one or more selected desirable characteristicsor which exhibit the desired characteristic(s) when in hybridcombination. Examples of desirable traits may include, in specificembodiments, high seed yield, high seed germination, seedling vigor,high fruit yield, disease tolerance or resistance, and adaptability forsoil and climate conditions. Consumer-driven traits, such as a fruitshape, color, texture, and taste are other examples of traits that maybe incorporated into new lines of spinach plants developed by thisinvention.

C. Further Embodiments of the Invention

In certain aspects of the invention, plants described herein areprovided modified to include at least a first desired heritable trait.Such plants may, in one embodiment, be developed by a plant breedingtechnique called backcrossing, wherein essentially all of themorphological and physiological characteristics of a variety arerecovered in addition to a genetic locus transferred into the plant viathe backcrossing technique. The term single locus converted plant asused herein refers to those spinach plants which are developed by aplant breeding technique called backcrossing or by genetic engineering,wherein essentially all of the morphological and physiologicalcharacteristics of a variety are recovered or conserved in addition tothe single locus introduced into the variety via the backcrossing orgenetic engineering technique, respectively. By essentially all of themorphological and physiological characteristics, it is meant that thecharacteristics of a plant are recovered or conserved that are otherwisepresent when compared in the same environment, other than an occasionalvariant trait that might arise during backcrossing, introduction of atransgene, or application of a genetic engineering technique.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentalspinach plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental spinach plant towhich the locus or loci from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a spinach plant isobtained wherein essentially all of the morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred locus from the nonrecurrentparent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered and the genetic distance between the recurrentand nonrecurrent parents. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele, or an additive allele (between recessive anddominant), may also be transferred. In this instance it may be necessaryto introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

In one embodiment, progeny spinach plants of a backcross in which aplant described herein is the recurrent parent comprise (i) the desiredtrait from the non-recurrent parent and (ii) all of the physiologicaland morphological characteristics of spinach the recurrent parent asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

New varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

With the development of molecular markers associated with particulartraits, it is possible to add additional traits into an established germline, such as represented here, with the end result being substantiallythe same base germplasm with the addition of a new trait or traits.Molecular breeding, as described in Moose and Mumm, 2008 (PlantPhysiol., 147: 969-977), for example, and elsewhere, provides amechanism for integrating single or multiple traits or QTL into an eliteline. This molecular breeding-facilitated movement of a trait or traitsinto an elite line may encompass incorporation of a particular genomicfragment associated with a particular trait of interest into the eliteline by the mechanism of identification of the integrated genomicfragment with the use of flanking or associated marker assays. In theembodiment represented here, one, two, three or four genomic loci, forexample, may be integrated into an elite line via this methodology. Whenthis elite line containing the additional loci is further crossed withanother parental elite line to produce hybrid offspring, it is possibleto then incorporate at least eight separate additional loci into thehybrid. These additional loci may confer, for example, such traits as adisease resistance or a fruit quality trait. In one embodiment, eachlocus may confer a separate trait. In another embodiment, loci may needto be homozygous and exist in each parent line to confer a trait in thehybrid. In yet another embodiment, multiple loci may be combined toconfer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,herbicide resistance, resistance to bacterial, fungal, or viral disease,insect resistance, modified fatty acid or carbohydrate metabolism, andaltered nutritional quality. These comprise genes generally inheritedthrough the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. For this selection process, the progeny of the initialcross are assayed for viral resistance or the presence of thecorresponding gene prior to the backcrossing. Selection eliminates anyplants that do not have the desired gene and resistance trait, and onlythose plants that have the trait are used in the subsequent backcross.This process is then repeated for all additional backcross generations.

Selection of spinach plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection are well known in the art. Suchmethods will be of particular utility in the case of recessive traitsand variable phenotypes, or where conventional assays may be moreexpensive, time consuming, or otherwise disadvantageous. In addition,marker assisted selection may be used to identify plants comprisingdesirable genotypes at the seed, seedling, or plant stage, to identifyor assess the purity of a cultivar, to catalog the genetic diversity ofa germplasm collection, and to monitor specific alleles or haplotypeswithin an established cultivar.

Types of genetic markers which could be used in accordance with theinvention include, but are not necessarily limited to, Simple SequenceLength Polymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In particular embodiments of the invention, marker assisted selection isused to increase the efficiency of a backcrossing breeding scheme forproducing a spinach line comprising a desired trait. This technique iscommonly referred to as marker assisted backcrossing (MABC). Thistechnique is well-known in the art and may involve, for example, the useof three or more levels of selection, including foreground selection toidentity the presence of a desired locus, which may complement orreplace phenotype screening protocols; recombinant selection to minimizelinkage drag; and background selection to maximize recurrent parentgenome recovery.

D. Plants Derived by Genetic Engineering

Various genetic engineering technologies have been developed and may beused by those of skill in the art to introduce traits in plants. Incertain aspects of the claimed invention, traits are introduced intospinach plants via altering or introducing a single genetic locus ortransgene into the genome of a recited variety or progenitor thereof.Methods of genetic engineering to modify, delete, or insert genes andpolynucleotides into the genomic DNA of plants are well-known in theart.

In specific embodiments of the invention, improved spinach lines can becreated through the site-specific modification of a plant genome.Methods of genetic engineering include, for example, utilizingsequence-specific nucleases such as zinc-finger nucleases (see, forexample, U.S. Pat. Appl. Pub. No. 2011-0203012); engineered or nativemeganucleases; TALE-endonucleases (see, for example, U.S. Pat. Nos.8,586,363 and 9,181,535); and RNA-guided endonucleases, such as those ofthe CRISPR/Cas systems (see, for example, U.S. Pat. Nos. 8,697,359 and8,771,945 and U.S. Pat. Appl. Pub. No. 2014-0068797). One embodiment ofthe invention thus relates to utilizing a nuclease or any associatedprotein to carry out genome modification. This nuclease could beprovided heterologously within donor template DNA for templated-genomicediting or in a separate molecule or vector. A recombinant DNA constructmay also comprise a sequence encoding one or more guide RNAs to directthe nuclease to the site within the plant genome to be modified. Furthermethods for altering or introducing a single genetic locus include, forexample, utilizing single-stranded oligonucleotides to introduce basepair modifications in a spinach plant genome (see, for example Sauer etal., Plant Physiol, 170(4):1917-1928, 2016).

Methods for site-directed alteration or introduction of a single geneticlocus are well-known in the art and include those that utilizesequence-specific nucleases, such as the aforementioned, or complexes ofproteins and guide-RNA that cut genomic DNA to produce a double-strandbreak (DSB) or nick at a genetic locus. As is well-understood in theart, during the process of repairing the DSB or nick introduced by thenuclease enzyme, a donor template, transgene, or expression cassettepolynucleotide may become integrated into the genome at the site of theDSB or nick. The presence of homology arms in the DNA to be integratedmay promote the adoption and targeting of the insertion sequence intothe plant genome during the repair process through homologousrecombination or non-homologous end joining (NHEJ).

In another embodiment of the invention, genetic transformation may beused to insert a selected transgene into a plant of the invention ormay, alternatively, be used for the preparation of transgenes which canbe introduced by backcrossing. Methods for the transformation of plantsthat are well-known to those of skill in the art and applicable to manycrop species include, but are not limited to, electroporation,microprojectile bombardment, Agrobacterium-mediated transformation, anddirect DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

An efficient method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, particles arecoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target cells. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates.Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Nat. Biotechnol., 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Nat. Biotechnol., 3:629-635, 1985; U.S.Pat. No. 5,563,055).

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, for example,Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al.,Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986;Marcotte et al., Nature, 335:454, 1988). Transformation of plants andexpression of foreign genetic elements is exemplified in Choi et al.(Plant Cell Rep., 13:344-348, 1994), and Ellul et al. (Theor. Appl.Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for plant gene expressioninclude, but are not limited to, the cauliflower mosaic virus (CaMV)P-35S promoter, which confers constitutive, high-level expression inmost plant tissues (see, for example, Odel et al., Nature, 313:810,1985), including in monocots (see, for example, Dekeyser et al., PlantCell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389,1990); a tandemly duplicated version of the CaMV 35S promoter, theenhanced 35S promoter (P-e35S); the nopaline synthase promoter (An etal., Plant Physiol., 88:547, 1988); the octopine synthase promoter(Fromm et al., Plant Cell, 1:977, 1989); the figwort mosaic virus(P-FMV) promoter as described in U.S. Pat. No. 5,378,619; an enhancedversion of the FMV promoter (P-eFMV) where the promoter sequence ofP-FMV is duplicated in tandem; the cauliflower mosaic virus 19Spromoter; a sugarcane bacilliform virus promoter; a commelina yellowmottle virus promoter; and other plant virus promoters known to expressin plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, or developmental signals can also beused for expression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat (Callis et al., Plant Physiol., 88:965,1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., PlantCell, 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell,3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpson etal., EMBO J., 4:2723, 1985), (3) hormones, such as abscisic acid(Marcotte et al., Plant Cell, 1:969, 1989), (4) wounding (e.g., wunl,Siebertz et al., Plant Cell, 1:961, 1989); or (5) chemicals, such asmethyl jasmonate, salicylic acid, or Safener. It may also beadvantageous to employ organ-specific promoters (e.g., Roshal et al.,EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988;Bustos et al., Plant Cell, 1:839, 1989).

Exemplary nucleic acids which may be introduced to plants of thisinvention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a spinach plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a spinach plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see, for example, Gibson and Shillito, Mol.Biotech., 7:125, 1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

E. Definitions

In the description and table herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a genetic locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions or transgenes from one geneticbackground into another.

Crossing: The mating of two parent plants.

Cross-Pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent. Marker: A readily detectable phenotype,preferably inherited in codominant fashion (both alleles at a locus in adiploid heterozygote are readily detectable), with no environmentalvariance component, i.e., heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Resistance: As used herein, the terms “resistance” and “tolerance” areused interchangeably to describe plants that show no symptoms to aspecified biotic pest, pathogen, abiotic influence, or environmentalcondition. These terms are also used to describe plants showing somesymptoms but that are still able to produce marketable product with anacceptable yield. Some plants that are referred to as resistant ortolerant are only so in the sense that they may still produce a crop,even though the plants are stunted and the yield is reduced.

Regeneration: The development of a plant from tissue culture.

Royal Horticultural Society (RHS) Color Chart Value: The RHS Color Chartis a standardized reference which allows accurate identification of anycolor. A color's designation on the chart describes its hue, brightness,and saturation. A color is precisely named by the RHS Color Chart byidentifying the group name, sheet number, and letter, e.g.,Yellow-Orange Group 19A or Red Group 41B.

Self-Pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing or genetic engineering ofa locus, wherein essentially all of the morphological and physiologicalcharacteristics of a spinach variety are recovered in addition to thecharacteristics of the single locus.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a spinach plant by transformation orsite-specific modification.

F. Deposit Information

A deposit of at least 625 seeds of spinach line SMBS013-1209F, disclosedabove and recited in the claims, has been made with theProvasoli-Guillard National Center for Marine Algae and Microbiota(NCMA), 60 Bigelow Drive, East Boothbay, Me., 04544 USA. The date ofdeposit for those deposited seeds of spinach line SMBS013-1209F is May28, 2020. The accession number for those deposited seeds of spinach lineSMBS013-1209F is NCMA Accession No. 202005167. Upon issuance of apatent, all restrictions upon the deposit will be removed, and thedeposit is intended to meet all of the requirements of 37 C.F.R. §§1.801-1.809. The deposit has been accepted under the Budapest Treatywill be maintained in the depository for a period of 30 years, 5 yearsafter the last request, or the effective life of the patent, whicheveris longer, and will be replaced if necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

What is claimed:
 1. A spinach plant comprising at least a first set ofchromosomes of spinach line SMBS013-1209F, a sample of seed of said linehaving been deposited under NCMA Accession No.
 202005167. 2. A spinachseed that produces the plant of claim
 1. 3. The plant of claim 1,wherein the plant is a plant of said spinach line SMBS013-1209F.
 4. Theplant of claim 1, wherein the plant is a hybrid plant.
 5. The seed ofclaim 2, wherein said seed is a seed of said spinach line SMBS013-1209F.6. The seed of claim 2, wherein the seed is a seed of a hybrid plant. 7.A plant part of the plant of claim 1, wherein said plant part comprisesa cell of said plant, and wherein said cell comprises at least the firstset of chromosomes of spinach line SMBS013-1209F.
 8. A spinach planthaving all of the physiological and morphological characteristics of theplant of claim
 1. 9. A tissue culture of regenerable cells of the plantof claim 1, wherein said tissue culture comprises a cell of said plant,and wherein said cell comprises at least the first set of chromosomes ofSMBS013-1209F.
 10. A method of vegetatively propagating a spinach plantcomprising at least a first set of chromosomes of spinach lineSMBS013-1209F, the method comprising the steps of: (a) collecting tissuecapable of being propagated from the plant of claim 1; and (b)propagating a spinach plant from said tissue.
 11. A method ofintroducing a trait into a spinach line, the method comprising: (a)utilizing as a recurrent parent the plant of claim 3 by crossing saidplant with a donor plant that comprises a trait to produce F₁ progeny;(b) selecting an F₁ progeny that comprises the trait; (c) backcrossingthe selected F₁ progeny with a plant of the same line used as therecurrent parent in step (a) to produce backcross progeny; (d) selectinga backcross progeny that comprises the trait and otherwise comprises allof the morphological and physiological characteristics of the recurrentparent line used in step (a); and (e) repeating steps (c) and (d) threeor more times to produce a selected fourth or higher backcross progeny.12. A spinach plant produced by the method of claim 11, wherein saidplant comprises the trait and otherwise comprises all of themorphological and physiological characteristics of spinach lineSMBS013-1209F.
 13. A method of producing a spinach plant comprising anadded trait, the method comprising introducing a transgene conferringthe trait into the plant of claim
 1. 14. A spinach plant produced by themethod of claim
 13. 15. A spinach plant comprising at least a first setof chromosomes of spinach line SMBS013-1209F, a sample of seed of saidline having been deposited under NCMA Accession No. 202005167, furthercomprising a transgene.
 16. The plant of claim 15, wherein saidtransgene confers a trait selected from the group consisting of malesterility, herbicide tolerance, insect resistance, pest resistance,disease resistance, modified fatty acid metabolism, environmental stresstolerance, modified carbohydrate metabolism, and modified proteinmetabolism.
 17. A spinach plant comprising at least a first set ofchromosomes of spinach line SMBS013-1209F, a sample of seed of said linehaving been deposited under NCMA Accession No. 202005167, furthercomprising a single locus conversion.
 18. The plant of claim 17, whereinsaid single locus conversion confers a trait selected from the groupconsisting of male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, modified fatty acid metabolism,environmental stress tolerance, modified carbohydrate metabolism, andmodified protein metabolism.
 19. A method for producing a seed of aspinach plant derived from spinach line SMBS013-1209F, the methodcomprising the steps of: (a) crossing the plant of claim 1 with itselfor a different spinach plant; and (b) allowing a seed of a spinach lineSMBS013-1209F-derived spinach plant to form.
 20. A method of producing aseed of a spinach line SMBS013-1209F-derived spinach plant, the methodcomprising the steps of: (a) producing a spinach lineSMBS013-1209F-derived spinach plant from a seed produced by crossing theplant of claim 1 with itself or a different spinach plant; and (b)crossing the spinach line SMBS013-1209F-derived spinach plant withitself or a different spinach plant to obtain a seed of a furtherspinach line SMBS013-1209F-derived spinach plant.
 21. The method ofclaim 20, the method further comprising repeating said producing andcrossing steps of (a) and (b) using the seed from said step (b) forproducing the plant according to step (a) for at least one generation toproduce a seed of an additional spinach line SMBS013-1209F-derivedspinach plant.
 22. A method of producing food, the method comprising:(a) obtaining the plant of claim 1, wherein the plant has beencultivated to maturity; and (b) collecting a plant part capable of beingused as food from said plant.